This HIVRAD program merges the complementary expertise of leading HIV-1 researchers in order to provide a fundamental advance towards an HIV-1 vaccine than can elicit broadly neutralizing antibodies (NAbs). This innovative program comprises two Research Projects: HIV-1 Env Vaccine Design (Dr. John Moore, Cornell University) and HIV-1 Trimer Crystallography (Dr. Ian Wilson, The Scripps Research Institute). The Projects are supported by scientific and administrative Cores (Dr. William Olson, Progenies Pharmaceuticals). This program leverages our recent successes in generating stable, proteolytically mature gp140 trimers (SOSIP gp140s) that mimic virion-associated Env in topology and antigenicity. As immunogens, SOSIP trimers have proven to be superior to matched gp120s in eliciting NAbs in animals. In the HIVRAD, the SOSIP template will be tailored for structural studies and for further improvements in immunogenicity in animals. Our overall goals of the HIVRAD are reflected in three major milestones: 1) determine the structure of cleaved env trimers at <4A resolution, 2) demonstrate methods to overcome HIV-1 Env's immunosuppressive properties, and 3) identify a SOSIP trimer vaccine that elicits heterologous neutralization of diverse HIV-1 isolates. Given the present rudimentary knowledge of trimer structure, an atomic-level view has the potential to spur development of a new generation of rationally designed Env vaccines, and our preliminary studies on crystallizing SOSIP trimers provide high enthusiasm and guarded optimism for success. Parallel studies will evaluate SOSIP trimers that have been modified so as to improve presentation of NAb epitopes and/or remove Env immunosuppressive features. Animal immunogenicity studies will be complemented with in vitro studies that compare modified and unmodified forms of Env for their ability to elicit immunosuppressive responses in human DCs. In addition to the primary Projects and Cores, the HIVRAD includes additional leading academic and corporate collaborators who lend specialized expertise in the areas of vaccine delivery (Aldevron, Inc.), NAb analyses (Monogram Biosciences), NAb specificity analyses (Dr. James Binley), in vitro immunogenicity studies (VaxDesign), and exploratory immunogenetics (Dr. Sunil Ahuja). Our shared goal is to overcome key structural and immunological challenges to developing a successful Env-based HIV-1 vaccine. PROJECT 1: HIV-1 Env Vaccine Design (Moore, J) PROJECT 1 DESCRIPTION (provided by applicant): The goal of Project 1 of the HIVRAD is to design vaccines intended to induce NAbs, based on accumulated and emerging knowledge of structure-function relationships within the HIV-1 Env complex and of how Env proteins interact with cells of the immune system, in vitro and in vivo. We propose three inter-related Specific Aims. Aim 1: To further modify the amino acid sequence of HFV-1 envelope glycoproteins. to create novel forms that improve the immunogenicity of neutralization epitopes and facilitate structural studies. We will build on the SOSIP method for making stable, cleaved gp!40 trimers, by identifying and testing additional substitutions that stabilize gp41-gp41 interactions, by introducing other sequence changes into gp!20 and/or gp41 that are intended to create or better expose NAb epitopes, and by eliminating immunosuppressive regions of the Env complex. The latter studies will be coordinated with research outlined in Aim 2, and will lead to the creation of new immunogens for testing in small animals in Aim 3, and also in the MIMIC system within Core B. We will also make modifications to Env trimers that are intended to reduce protein heterogeneity and/or flexibility, and thereby facilitate X-ray crystallography studies to be conducted by Ian Wilson (Project 2). Aim 2: To study in vitro how to overcome immunosuppressive effects of gp120 by the use of adjuvants and TLR activators. We will study the immunosuppressive effects of the mannose moieties of gp120 glycans in vitro, using both human and murine cell-based systems, particularly dendritic cells (DCs). The abilities of TLR activators and other adjuvant-associated molecules to overcome these adverse effects will then be tested. We will, in addition, collaborate with the groups headed by Eric Mishkin and Sunil Ahuja, to derive additional immunologic and genetic information using the VaxDesign MIMIC system (Core B). The outcome of these experiments will facilitate the construction of new immunogens in Aim 1, and the rational design of immunization regimens in Aim 3 and Core B that are intended to maximize the antibody response to Env. Aim 3: To evaluate the immunogenicity of modified Env trimers in small animals. We will design and evaluate rabbit and mouse immunization studies that will be conducted within Core B, to determine whether the various modifications to Env glycoproteins that we identified and evaluated in Aims 1 and 2, can alter the immune response to these and co-administered antigens. We will also compare the immunogenicity of Envproteins with other antigens, alone and co-administered, to determine whether Env proteins can suppress or modify immune responses, including the IgG subclass pattern, to themselves and other antigens (HTV-1 derived or unrelated). The design of some experiments will also take into account information on adjuvants and TLR activators generated in Aim 2 and by using the VaxDesign MIMIC system in Core B. In an iterative process, the immunization experiments will themselves guide the design of additional studies to be carried out by VaxDesign using the MIMIC system (see Core B). The overall outcome of these various studies will help design additional Env sequence modifications in Aim 1 that are intended to further improve immunogenicity or eliminate the causes of immunosuppression.